Cell migration is a complex process that involves reorganization of the cytoskeleton, protrusion of a leading edge, attachment to the matrix, development of contractile force for movement, and detachment of cell surface contacts. There is dynamic and extensive reorganization of the cytoskeleton to accomplish netmigration. The involvement of actomyosin cellular myofilaments in migration is generally accepted, however, the specific functions of actomyosin in time and space are not well understood, and are the focus of this project. Myosin LC phosphorylation is a biochemical marker for contractile activity and the activity of the myosin phosphatase MLCP has a major role in determining the levels of LC phosphorylation. Thus, considerable' attention has been drawn to mechanisms for regulation of the MLCK. One pathway involves the small GTPase rho, and rho-activated kinase, which inactivates MLCP by phosphorylating the M130 regulatory subunit. Another pathway involves a heat stable protein called CPI-17 purified from smooth muscle, which is a potent and highly specific inhibitor of MLCP, but only after phosphorylation by protein kinase C at a Thr residue in the sequence RVTVK. In other cells the mRNA of an analogue of CPI- 17 (called PNG) is expressed, but the protein has not' been biochemically characterized and nothing is known about its phosphorylation or function. Under Aim 1 this PNG protein will be cloned, expressed and studied in parallel with CPI. Together, the CPI and PNG proteins are, referred to as myosin phosphatase inhibitor proteins, MPIP. The sites involved in binding MPIP to MLCP will be' mapped under Aim 2. Overexpression of CPI in fibroblasts inhibits cell spreading, focal adhesion formation and elevates LC20 phosphorylation. Under Aim 3, steps in cytoskeletal reorganization and motility that are being affected by MPIP expression will be examined. How different receptors signal through MPIP compared to rho, to inhibit MLCP and activate actomyosin will be tested under Aim 4. Expression of constituitively active and dominant negative forms GTPases such as rho, rac, cdc42 and TC10 will be used to examine signaling through MPIPs, also under Aim 4. One of the isoforms of the catalytic subunit of PP1 that purifies as a subunit of MLCP also specifically associates with focal adhesions and co-immunoprecipitates with either focal adhesion kinase or integrin. The structural basis for this targeting will be examined under Aim 5 with the expectation that protein antagonists of targeting might be produced to reveal the functional importance of this specific association. Overall, this project explores the unknown and underappreciated role of type-1 protein phosphatase in the process of cytoskeletal reorganization and cell migration.